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Una fenilalanina en el sitio activo dirige la unión al sustrato y la escisión de C-H en la dioxigenasa TauD,
Kevin P McCusker1, Judith P Klinman
1Department of Chemistry, California Institute for Quantitative Biosciences, University of California, Berkeley, California 94720, USA.
Journal of the American Chemical Society
|March 23, 2010
Resumen
La fenilalanina 159 en la taurina alfa-cetoglutarato dioxigenasa (TauD) es crucial para la unión al sustrato y la escisión del enlace C-H. Las mutaciones interrumpen las distancias óptimas, lo que afecta la eficiencia de las enzimas y la superposición de la función de onda hidrogénica.
Área de la Ciencia:
- La bioquímica es la bioquímica.
- Enzimología Enzimología.
- Ingeniería de proteínas Ingeniería de proteínas.
Sus antecedentes:
- Las enzimas que escinden enlaces C-H a menudo dependen de núcleos hidrofóbicos para posicionar sustratos para la catálisis.
- La taurina alfa-cetoglutarato dioxigenasa (TauD) utiliza un sitio activo de la especie Fe ((IV) = O para la oxidación de enlaces C-H.
- El residuo F159 en TauD está implicado en la unión al sustrato y la orientación para la transferencia del átomo de hidrógeno.
Objetivo del estudio:
- Para investigar el papel del residuo F159 en el mecanismo catalítico de TauD.
- Para determinar cómo las mutaciones en F159 afectan la unión al sustrato, la escisión de enlaces C-H y la activación del oxígeno.
- Para explorar la relación entre la estructura del sitio activo, la orientación del sustrato y la eficiencia enzimática.
Principales métodos:
- Mutagenesis dirigida al sitio de F159 en TauD a residuos hidrofóbicos más pequeños (L, V, A).
- Estudios de cinética enzimática bajo condiciones de rotación de estado estacionario.
- Extracción de los efectos del isótopo cinético intrínseco del deuterio (KIEs) utilizando transitorios de un solo volumen de negocios.
Principales resultados:
- Las mutaciones en F159 redujeron significativamente las tasas de unión al sustrato y de escisión de los enlaces C-H.
- La contribución de la etapa química a k ((cat) aumentó en los mutantes F159, lo que indica cambios en las etapas que limitan la velocidad.
- Los KIEs intrínsecos de deuterio aumentaron de 2 a 4 veces para F159V en comparación con el tipo silvestre y F159L.
- Se observó un cambio distinto en el comportamiento de la enzima entre las mutaciones F159L y F159V.
Conclusiones:
- El residuo F159 es crítico para mantener la distancia internuclear óptima entre el sustrato y el sitio activo Fe(IV) = O.
- La pérdida de un solo grupo metileno en F159V interrumpe la capacidad del sitio activo para facilitar la transferencia eficiente de átomos de hidrógeno.
- Los hallazgos apoyan un modelo modular de sitio activo para TauD, con roles funcionales distintos para la unión/orientación del sustrato y la activación del oxígeno.
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